Herpes Simplex Virus Type 1 Entry Research Articles

Plant Cell-Engineered Gold Nanoparticles Conjugated to Quercetin Inhibit SARS-CoV-2 and HSV-1 Entry.

Recent studies have revealed considerable promise in the antiviral properties of metal nanomaterials, specifically when biologically prepared. This study demonstrates for the first time the antiviral roles of the plant cell-engineered gold nanoparticles (pAuNPs) alone and when conjugated with quercetin (pAuNPsQ). We show here that the quercetin conjugated nanoparticles (pAuNPsQ) preferentially inhibit the cell entry of two medically important viruses-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and herpes simplex virus type-1 (HSV-1) using different mechanisms. Interestingly, in the case of SARS-CoV-2, the pre-treatment of target cells with pAuNPsQ inhibited the viral entry, but the pre-treatment of the virus with pAuNPsQ did not affect viral entry into the host cell. In contrast, pAuNPsQ demonstrated effective blocking capabilities against HSV-1 entry, either during the pre-treatment of target cells or by inducing virus neutralization. In addition, pAuNPsQ also significantly affected HSV-1 replication, evidenced by the plaque-counting assay. In this study, we also tested the chemically synthesized gold nanoparticles (cAuNPs) of identical size and shape and observed comparable effects. The versatility of plant cell-based nanomaterial fabrication and its modification with bioactive compounds opens a new frontier in therapeutics, specifically in designing novel antiviral formulations.

Antiviral Activity of Oridonin Against Herpes Simplex Virus Type 1.

In search of new potent treatment of herpes simplex keratitis (HSK), inhibitory effect of oridonin (Ori) on herpes simplex virus type 1 (HSV-1) was validated by experiments. For evaluating inhibitory effect of oridonin on herpes simplex virus type 1, a series of in-vivo and in-vitro studies were carried out. Mouse HSV-1 infection model was used in the in-vivo experiments. Experimental mice were classified infivedifferentgroups: Mock (mock-infected), HSV-1+ DMSO, HSV-1+ Ori, HSV-1+ ACV, combined Ori and ACV+HSV-1. Corneas of Mock, HSV-1+ DMSO, HSV-1+ Ori group were sent formRNA-sequencing after 3 days post infection (dpi). The expression of virus and host-related genes was evaluated by quantitative real-time polymerase chain reaction (qPCR). Vero cells HSV-1 infection models were used in the in-vitro experiments. The application of ACV, Oridonin alone or a combination of both could alleviate HSV-1 severity and inhibit HSV-1 virus replication in C57BL/6 mice models. qPCR showed that compared with mock group, the expression of interleukin-6 (il-6), interleukin-1α (il-1α), and Tumor-necrosis factor-alpha (tnf-α) was up-regulated in DMSO+HSV-1 group and suppressed in other three group. Moreover, the expression of nod-like receptor protein (nlrp3), caspase 1 and interleukin-1β (il-1β) were depressed in the oridonin-treated group. Oridonin significantly inhibits HSV-1 replication, HSV-1 related gene expression, and the production of progeny HSV-1 viruses in vitro. Besides, oridonin affect the replication phase but not HSV-1 entry or penetration and cannot inactivate HSV-1. Oridonin alleviates herpes simplex keratitis infection in mouse, which may be attributed to inhibition of the NLRP3-inflammasome-IL-1β pathway. Our study illustrates that Oridonin has potential promise for application in treating HSK and other diseases caused by HSV-1 infection.

Inhibitory activity and mechanism of silver nanoparticles against herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1) is a common pathogen that infects 50-90% of the world's population and causes a variety of diseases, some of which can be life-threatening. Silver nanoparticles (AgNPs) have been shown to have broad-spectrum antiviral activity. In this study, we investigated the activity of AgNPs against HSV-1 and found that AgNPs effectively inhibited plaque formation and HSV-1 progeny production, reduced the genomic load, and interfered with HSV-1 mRNA expression and protein synthesis. Transmission electron microscopy showed that AgNPs interacted with HSV-1 and altered the shape of the viral particles. Furthermore, AgNPs affected the entry of HSV-1 into cells as well as their release and cell-to-cell spread. AgNPs were also found to downregulate the expression of pro-inflammatory cytokines upon HSV-1 infection. Combined treatment with AgNPs and acyclovir (ACV) confirmed that AgNPs significantly enhanced the inhibitory effect of ACV against HSV-1. Our findings may contribute to an understanding of the mechanism of the antiviral effect of AgNPs against HSV-1 and help to provide a theoretical basis for their clinical application.

Plasma Membrane-Derived Liposomes Exhibit Robust Antiviral Activity against HSV-1.

Plasma membranes host a plethora of proteins and glycans on their outer surface that are exploited by viruses to enter the cells. In this study, we have utilized this property to limit a viral infection using plasma membrane-derived vesicles. We show that plasma membrane-derived liposomes are prophylactically and therapeutically competent at preventing herpes simplex virus type-1 (HSV-1) infection. Plasma membrane liposomes derived from human corneal epithelial (HCE) cells, which are natural targets of HSV-1 infection, as well as Vero and Chinese hamster ovary (CHO) cells were used in this study. Our study clearly demonstrates that HCE and Vero-derived cellular liposomes, which express the viral entry-specific cell surface protein receptors, exhibit robust antiviral activity especially when compared to CHO-derived liposomes, which lack the relevant HSV-1 entry receptors. Further experimentation of the plasma membrane-derived liposomes with HSV type-2 (HSV-2) and pseudorabies virus yielded similar results, indicating strong potential for the employment of these liposomes to study viral entry mechanisms in a cell free-environment.

Go go gadget glycoprotein!: HSV-1 draws on its sizeable glycoprotein tool kit to customize its diverse entry routes.

All viruses must enter cells to replicate [1]. Entry is thus the first hurdle a virus must overcome for a successful infection. Given the astonishing diversity of viruses that infect mammals alone—hundreds of thousands of different viruses according to some estimates [2]—it is unsurprising that their entry routes into cells are just as diverse. A major structural distinction that dictates the entry mode is the presence or absence of a lipid bilayer surrounding the viral nucleocapsid, which defines a virus as enveloped or nonenveloped, respectively. Nonenveloped viruses enter cells by endocytosis and subsequently penetrate the endosomal membrane by a variety of mechanisms including pore formation and endosomal fragmentation due to disruptive changes in membrane curvature [3], whereas all enveloped viruses must fuse their envelope with a host cell membrane: either the plasma membrane or the membrane of the endosomal vesicle following cellular uptake [4]. Regardless of the entry route, all viruses initially attach to the surface of the host cell by binding a cellular receptor. After attachment, enveloped viruses must employ fusogens—specialized viral surface glycoproteins that mediate the merger of the viral and host membranes by bringing them together as they undergo large, energetically favorable conformational changes. To do this, a spring-loaded fusogen must be triggered once the virus arrives at the right cell and/or the right intracellular compartment (such as an endosome, for example), either by binding a receptor (or a coreceptor) or by sensing the acidic pH of the endosome [4]. In many enveloped viruses, the receptor-binding and the fusogenic functions are mediated by different domains of a single glycoprotein. For example, the human immunodeficiency virus (HIV) envelope protein, Env, the sole glycoprotein encoded by HIV, binds the cellular glycoprotein cluster of differentiation 4 (CD4) and a coreceptor, C-X-C chemokine receptor 4 (CXCR4) or C-C chemokine receptor 5 (CCR5), on the surface of CD4+ T cells and also serves as the fusogen [5]. The influenza virus glycoprotein hemagglutinin binds an attachment receptor, sialic acid, and undergoes low-pH-triggered fusogenic conformational changes upon endocytosis [6]. In some cases, for example, in paramyxoviruses, the receptor-binding and the fusogenic functions are mediated by separate glycoproteins, and the fusogen receives the triggering signal from the receptor-binding viral protein [7]. Most enveloped viruses thus contain multiple copies of only one or two glycoproteins, which mediate viral attachment and entry into target cells [5–15]. Yet, entry of herpesviruses—large enveloped viruses that infect a wide variety of cells—is more complex, as it requires multiple viral glycoproteins (typically, at least three) and diverse host receptors [16]. Moreover, the coordinated activity of these multiple viral glycoproteins permits entry into different cell types by different routes. Whereas in some herpesviruses, such as human cytomegalovirus (HCMV) or Epstein–Barr virus (EBV), the use of particular entry routes correlates with the involvement of specific viral glycoprotein complexes [17, 18], in other herpesviruses, notably, herpes simplex virus type 1 (HSV-1), the picture is less clear [19]. Nonetheless, the entry mechanisms of all herpesviruses into a given cell, and particularly, the selection of the entry route, are complex and incompletely understood. The HSV-1 replication cycle in humans necessitates the infection of different cell types, chiefly, epithelial and neuronal cells. Although it is known that HSV-1 enters these cells by different mechanisms—endocytosis (epithelial cells) and fusion at the plasma membrane (neurons) [20, 21]—knowledge regarding HSV-1 glycoprotein involvement in the entry route–selection process is minimal. This raises the following question: How does HSV-1 select a particular route to enter different cell types? Although the answer remains elusive, this Pearl will summarize the current understanding of HSV-1 entry strategies and the players involved.

A synthetic glycosaminoglycan mimetic blocks HSV-1 infection in human iris stromal cells

Herpes simplex virus type-1 (HSV-1) is a significant pathogen that affects vision by targeting multiple regions in the human eye including iris. Using a focused library of synthetic non-saccharide glycosaminoglycan mimetics (NSGMs), we identified sulfated pentagalloylglucoside (SPGG) as a potent inhibitor of HSV-1 entry and cell-to-cell spread in the primary cultures of human iris stromal (HIS) cells isolated from eye donors. Using in vitro β-galactosidase reporter assay and plaque reduction assay, SPGG was found to inhibit HSV-1 entry in a dosage-dependent manner (IC50 ∼6.0 μM). Interestingly, a pronounced inhibition in HSV-1 entry and spread was observed in HIS cells, or a cell line expressing specific gD-receptor, when virions were pre-treated with mimetics suggesting a possible interaction between SPGG and the HSV-1 glycoprotein. To examine the significance of gD-SPGG interaction, HIS cells were pretreated with SPGG, which showed a significant reduction in gD binding. Taken together, our results provide strong evidence of SPGG being a novel viral entry inhibitor against ocular HSV infection.

Targeting Herpes Simplex Virus-1 gD by a DNA Aptamer Can Be an Effective New Strategy to Curb Viral Infection.

Herpes simplex virus type 1 (HSV-1) is an important factor for vision loss in developed countries. A challenging aspect of the ocular infection by HSV-1 is that common treatments, such as acyclovir, fail to provide effective topical remedies. Furthermore, it is not very clear whether the viral glycoproteins, required for HSV-1 entry into the host, can be targeted for an effective therapy against ocular herpes in vivo. Here, we demonstrate that HSV-1 envelope glycoprotein gD, which is essential for viral entry and spread, can be specifically targeted by topical applications of a small DNA aptamer to effectively control ocular infection by the virus. Our 45-nt-long DNA aptamer showed high affinity for HSV-1 gD (binding affinity constant [Kd] = 50 nM), which is strong enough to disrupt the binding of gD to its cognate host receptors. Our studies showed significant restriction of viral entry and replication in both in vitro and ex vivo studies. In vivo experiments in mice also resulted in loss of ocular infection under prophylactic treatment and statistically significant lower infection under therapeutic modality compared to random DNA controls. Thus, our studies validate the possibility that targeting HSV-1 entry glycoproteins, such as gD, can locally reduce the spread of infection and define a novel DNA aptamer-based approach to control HSV-1 infection of the eye.

Mapping sites of herpes simplex virus type 1 glycoprotein D that permit insertions and impact gD and gB receptors usage

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) is one of four glycoproteins essential for HSV entry and cell fusion. The purpose of this study was to determine the plasticity of gD to tolerate insertion or deletion mutations and to construct an oncolytic HSV-1 that utilizes the disialoganglioside GD2 as a HSV-1 entry receptor. We found that the N-terminus of gD tolerates long insertions, whereas residues adjacent to the gD Ig-like V-type core tolerated shorter insertions (up to 15 amino acids), but not greater than 60 amino acids. Recombinant HSV-1 containing the ch14.18 single chain variable fragment (scFv) at the N-terminus of gD failed to mediate entry, even though the ch14.18 scFv-gD chimera Fc bound to neuroblastoma cells expressing GD2. Finally, we found that hyperfusogenic gB mutants enhanced fusion to a greater degree with the gB receptor the paired immunoglobulin-like type 2 receptor alpha (PILRα) than with gD receptors HVEM and nectin-1. Hyperfusogenic gB could restore the fusion function with PILRα when a gD constructed contained only the “profusion domain” (PFD), suggesting the hyperfusogenic form of gB may regulate fusion of PILRα via a novel mechanism through gH/gL and the gD PFD.

Induction of Multiple miR-200/182 Members in the Brains of Mice Are Associated with Acute Herpes Simplex Virus 1 Encephalitis.

Important roles of microRNAs (miRNAs) in regulating the host response during viral infection have begun to be defined. However, little is known about the functional roles of miRNAs within an in vivo acute viral encephalitis model. We therefore identified global changes in miRNA expression during acute herpes simplex virus type 1 (HSV-1) encephalitis (HSVE) in mice. We found that many of the highly upregulated miRNAs (miR-155, miR-146a and miR-15b) detected in HSV-1 infected brain tissue are known regulators of inflammation and innate immunity. We also observed upregulation of 7 members belonging to the related group of miRNAs, the miR-200 family and miR-182 cluster (miR-200/182). Using in situ hybridization, we found that these miRNAs co-localized to regions of the brain with severe HSVE-related pathology and were upregulated in various cell types including neurons. Induction was apparent but not limited to cells in which HSV-1 was detected by immunohistochemistry, suggesting possible roles of these miRNAs in the host response to viral-induced tissue damage. Bioinformatic prediction combined with gene expression profiling revealed that the induced miR-200/182 members could regulate the biosynthesis of heparan sulfate proteoglycans. Using luciferase assays, we found that miR-96, miR-141, miR-183 and miR-200c all potentially targeted the syndecan-2 gene (Sdc2), which codes for a cell surface heparan sulfate proteoglycan involved in HSV-1 cellular attachment and entry.

Distribution of cellular HSV-1 receptor expression in human brain

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

Abstract 2351: Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models

Abstract Background: Talimogene laherparepvec is an oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1), designed to kill neoplastic cells through two mechanisms: a) direct lysis and b) stimulation of a tumor antigen-specific adaptive immune response by way of GM-CSF expression. In order to support mechanism of action studies in a mouse melanoma model relevant to talimogene laherparepvec's approved U.S. indication, we expressed the HSV-1 entry receptor Nectin1 on B16F10 melanoma cells. B16F10 cells have previously been shown to be resistant to HSV-1 due to lack of entry receptor expression (Miller et al., Mol Ther, 2001). The goal of our work was to determine if mNectin1 expression in B16F10 melanoma cells renders them sensitive to talimogene laherparepvec treatment. For preclinical studies we used OncoVEXmGM-CSF, an HSV-1 modified similarly to talimogene laherparepvec except that murine GM-CSF is expressed instead of human GM-CSF. Herein we describe studies to assess the efficacy of OncoVEXmGM-CSF in a novel B16F10-mNectin1 syngeneic melanoma model. Methods: Parental B16F10 cells were transduced with lentivirus expressing either mNectin1, or eGFP as a control. In vitro sensitivity was determined by plating both cell types in 96-well plates, infecting with serially diluted OncoVEXmGM-CSF and then measuring ATP 72 hours later. For efficacy studies, B16F10 or B16F10-mNectin1 cells were injected subcutaneously on the right flank of female C57BL/6 mice. On day 10, mice were randomized into treatment groups based on tumor volume (n = 10/group). Talimogene laherparepvec was delivered intratumorally (5×106 PFU/dose,) every 3 days during the first week. Tumor growth and body weight were measured twice per week throughout the experiment. Survival analysis was performed using a Kaplan-Meier estimator. Results: B16F10 cells were insensitive to OncoVEXmGM-CSF up to 100 MOI (multiplicity of infection). In contrast, two other syngeneic cell lines, A20 and CT-26, showed MOI IC50 of 1 and 20. B16F10 cells expressing mNectin1 showed high sensitivity to OncoVEXmGM-CSF with an MOI IC50 of 0.01. In contrast, B16F10 cells expressing eGFP were insensitive up to an MOI of 100. In vivo, B16F10 mNectin1 cells showed a similar growth pattern as the parental B16F10 when injected subcutaneously. However, OncoVEXmGM-CSF treatment of B16F10-mNectin1 tumors caused a highly significant (p<0.0001) inhibition of tumor growth and increase in median overall survival compared to the control group. B16F10 mNectin1 tumors that grew during or following OncoVEXmGM-CSF treatment were shown to maintain mNectin1 expression suggesting other mechanisms of OncoVEXmGM-CSF resistance were involved. Conclusion: Expression of mNectin 1 in the B16F10 melanoma cell line confers sensitivity to OncoVEXmGM-CSF in vitro and extends median overall survival of mice bearing B16F10-muNectin1 tumors. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Petia Mitchell, Yannick Bulliard, Pedro J. Beltran. Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2351.

Liposome-Mediated Herpes Simplex Virus Uptake Is Glycoprotein-D Receptor-Independent but Requires Heparan Sulfate.

Cationic liposomes are widely used to facilitate introduction of genetic material into target cells during transfection. This study describes a non-receptor mediated herpes simplex virus type-1 (HSV-1) entry into the Chinese hamster ovary (CHO-K1) cells that naturally lack glycoprotein D (gD)-receptors using a commercially available cationic liposome: lipofectamine. Presence of cell surface heparan sulfate (HS) increased the levels of viral entry indicating a potential role of HS in this mode of entry. Loss of viral entry in the presence of actin de-polymerizing or lysosomotropic agents suggests that this mode of entry results in the endocytosis of the lipofectamine-virus mixture. Enhancement of HSV-1 entry by liposomes was also demonstrated in vivo using a zebrafish embryo model that showed stronger infection in the eyes and other tissues. Our study provides novel insights into gD receptor independent viral entry pathways and can guide new strategies to enhance the delivery of viral gene therapy vectors or oncolytic viruses.

Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells.

Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus–unable to replicate in ICP4-defficient cells- showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells.

Heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin.

Although it is known that inhibitors of heat shock protein 90 (Hsp90) can inhibit herpes simplex virus type 1 (HSV-1) infection, the role of Hsp90 in HSV-1 entry and the antiviral mechanisms of Hsp90 inhibitors remain unclear. In this study, we found that Hsp90 inhibitors have potent antiviral activity against standard or drug-resistant HSV-1 strains and viral gene and protein synthesis are inhibited in an early phase. More detailed studies demonstrated that Hsp90 is upregulated by virus entry and it interacts with virus. Hsp90 knockdown by siRNA or treatment with Hsp90 inhibitors significantly inhibited the nuclear transport of viral capsid protein (ICP5) at the early stage of HSV-1 infection. In contrast, overexpression of Hsp90 restored the nuclear transport that was prevented by the Hsp90 inhibitors, suggesting that Hsp90 is required for nuclear transport of viral capsid protein. Furthermore, HSV-1 infection enhanced acetylation of α-tubulin and Hsp90 interacted with the acetylated α-tubulin, which is suppressed by Hsp90 inhibition. These results demonstrate that Hsp90, by interacting with acetylated α-tubulin, plays a crucial role in viral capsid protein nuclear transport and may provide novel insight into the role of Hsp90 in HSV-1 infection and offer a promising strategy to overcome drug-resistance.

Calcium-signal facilitates herpes simplex virus type 1 nuclear transport through slingshot 1 and calpain-1 activation

Herpes simplex virus type 1 (HSV-1) can establish its latency in neurons and is associated with virus-induced pathological neurodegeneration in the nervous system. Here we show that viral penetration-induced calcium release facilitated HSV-1 intracellular trafficking through activating slingshot 1 (SSH), a phosphatase regulating actin filament dynamics. More detailed studies revealed that phospholipase C gamma 1, and the inositol 1,4,5-trisphosphate receptor isoform 1 were required for SSH activation. Besides, calpain-1, a calcium-dependent cysteine protease, was involved in viral intracellular migration. These results may lead to new targets for antiviral therapy.

Zebrafish 3-O-Sulfotransferase-4 Generated Heparan Sulfate Mediates HSV-1 Entry and Spread

Rare modification of heparan sulfate (HS) by glucosaminyl 3-O sulfotransferase (3-OST) isforms generates an entry receptor for herpes simplex virus type-1 (HSV-1). In the zebrafish (ZF) model multiple 3-OST isoforms are differentially expressed. One such isoform is 3-OST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-OST-4 isoform for HSV-1 entry. Expression of ZF 3-OST-4 into resistant Chinese hamster ovary (CHO-K1) cells promoted susceptibility to HSV-1 infection. This entry was 3-O sulfated HS (3-OS HS) dependent as pre-treatment of ZF 3-OST-4 cells with enzyme HS lyases (heparinase II/III) significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-OST-4 along with ZF 3-OST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-OST-4 alone. The role of ZF-3-OST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-OST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-OST-4 mediated HSV-1 entry was inhibited by anti-3O HS G2 peptide. Taken together our results demonstrate a role for ZF 3-OST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.

Inhibition of Myosin Light Chain Kinase Can be Targeted for the Development of New Therapies against Herpes Simplex Virus Type-1 Infection

Herpes simplex virus type-1 (HSV-1) is the leading cause of infectious blindness worldwide. Through a multistep process, HSV-1 enters into naturally susceptible human corneal epithelial (HCE) cells where it establishes an optimal environment for viral replication and spread. HSV-1 employment of cytoskeletal proteins, kinases, and cell signalling pathways is crucial for the entry process. Here we demonstrate that non-muscle myosin IIA (NM-IIA) and/or a myosin activating kinase, myosin light chain kinase (MLCK), can be targeted for the development of new and effective therapies against HSV-1. HCE cells were incubated with MLCK inhibitors ML-7 and ML-9 and NM-IIA inhibitor blebbistatin. Following the application of inhibitors, HSV-1 entry and spread to neighbouring HCE cells was evaluated. Upon application of MLCK inhibitors ML-7 and ML-9 and NM-IIA inhibitor blebbistatin, HSV-1 entry into HCE cells was significantly decreased. Furthermore, dramatic impairment of glycoprotein-mediated membrane fusion was seen in cells treated with MLCK inhibitors, thus establishing a role for MLCK activation in cell-to-cell fusion and multinucleated syncytial cell formation. These results also indicate that the activation of motor protein NM-IIA by MLCK is crucial for cytoskeletal changes required for HSV-1 infection of corneal cells. We provide new evidence that NM-IIA and MLCK can be used as effective antiviral targets against ocular herpes.

Role of Filopodia in HSV-1 Entry into Zebrafish 3-O-Sulfotransferase-3-Expressing Cells

Heparan sulfate proteoglycans (HSPGs) modified by zebrafish (ZF) encoded glucosaminyl 3-O sulfotransferase-3 (3-OST-3) generate a receptor for herpes simplex virus type-1 (HSV-1) entry and spread. In order to elucidate the mechanism by which HSV-1 enters into ZF-3-OST-3 cells, we investigated the mode of viral entry. Under high resolution scanning electron microscopy (SEM), actin cytoskeleton changes were observed by a dramatic increase in the number of filopodia formed during early interactions of HSV-1 with the target cells. While the increase in number was common among all the infected cells, the highest numbers of filopodia was observed in cells expressing the 3-OST-3 modified form of heparan sulfate (HS) encoded either by human or ZF. The levels of viral infection and filopodia induction were reduced with the actin polymerization inhibitors, Cytochalasin-D and Lantriculin B, suggesting an important role for actin reorganization during ZF-3-OST-3 mediated HSV-1 entry. Supporting an interesting possibility of filopodia usage during HSV-1 spread, pre-treatment of cytochalasin D in ZF-3-OST-3 cells drastically reduced virus glycoprotein induced cell fusion. Taken together, our results provide new evidence on the involvement of filopodia during HSV-1 infection of ZF-3-OST-3 cells and confirm a role for modified heparan sulfate in cytoskeleton rearrangement during HSV-1 entry.

Zebrafish encoded 3-O-sulfotransferase-2 generated heparan sulfate serves as a receptor during HSV-1 entry and spread

Previously we reported the role of zebrafish (ZF) encoded glucosaminyl 3-O-sulfotransferase-3 (3-OST-3) isoform in assisting herpes simplex virus type-1 (HSV-1) entry and spread by generating an entry receptor to HSV-1 envelope glycoprotein D (gD). However, the ability of ZF encoded 3-OST-2 isoform to participate in HSV-1 entry has not been determined although it is predominantly expressed in ZF brain, a prime target for HSV-1 to infect and establish lifelong latency. Here we report the expression cloning of ZF encoded 3-OST-2 isoform and demonstrate HSV-1 entry into resistant Chinese hamster ovary (CHO-K1) cells expressing the clone. Additional significance of ZF encoded 3-OST-2 receptor was demonstrated using medically important isolates of HSV-1. In addition, interference to HSV-1 entry was observed upon co-expression of HSV-1 gD and ZF 3-OST-2. Similarly HSV-1 entry was significantly inhibited by the pre-treatment of cells with enzyme HS lyases (heparinase II/III). Finally, ZF-3-OST-2 expressing CHO-K1 was able to fuse with HSV-1 glycoprotein expressing cells suggesting their role in HSV-1 spread. Taken together our result demonstrates a role for ZF 3-OST-2 in HSV-1 pathogenesis.

Members of 3-O-Sulfotransferases (3-OST) Family: A Valuable Tool from Zebrafish to Humans for Understanding Herpes Simplex Virus Entry

The journey of many viruses to infect cells begins when the virus first binds to cell surface heparan sulfate (HS). The initial step of cell attachment or binding during herpes simplex virus type-1 (HSV-1) entry is mediated by envelope glycoprotein B (gB) and C (gC). The binding is followed by fusion between virus envelope and cell membrane during which HSV-1 glycoprotein D (gD) interacts with a modified form of HS know as 3-O-sulfated heparan sulfate (3-OS HS). The rare modification of 3-O-sulfation on HS chain is governed by enzymes known as 3-O-sulfotransferase (3-OST). Currently, there are seven isoforms of human 3-OSTs that have been identified, and with the exception of 3-OST-1, all other 3-OST isoforms allow HSV-1 entry and spread. Recently, the product of the zebrafish (ZF)-encoded 3-OST-3 was also recognized as a gD receptor, which mediates HSV-1 entry and cell-cell fusion similar to human 3-OST-3. Interestingly, the ZF system expresses multiple isoforms of 3-OST which could be very useful for studying the involvement of HS and 3-OS HS in virus tropism and virus-induced inflammation. In addition, therapeutic targeting of 3-OST generated HS is likely to bring about novel interventions against HSV-1. In this review we have taken a closer look at the potential of both human and ZF encoded 3-OSTs as valuable tools in HSV entry and inflammation studies.